Taxane derivatives having a pyridyl substituted C13 side chain, their preparation and their use as anti-tumor agents

ABSTRACT

The invention relates to pharmacologically active compounds that have the ability to influence the polymerisation and depolymerisation of tubulin. 
     A range of natural mitosis toxins are used as anti-tumor agents or are in the process of being clinically tested. There are various classes of such mitosis toxins that either demonstrate their cytotoxic action by inhibiting the polymerisation of microtubules in the spindle system (for example, Vinca alkaloids, colchicine) or achieve their cytotoxic action by a GTP-independent increase in the polymerisation of the tubulin and by preventing the depolymerisation of microtubules (for example, taxol, taxoters). 
     Owing to their physico-chemical properties, hitherto not understood, and as a result of the characteristics of neoplastic cells, mitosis toxins have a certain selectivity for tumor cells, but there still remains a not inconsiderable cytotoxicity towards non-transformed cells. The search for more selective compounds that are easier to manufacture and--like the taxane class of substances--are able to inhibit the depolymerisation of microtubules, had, surprisingly, led to the discovery of borneol esters, as described in P 4416374.6 and 19513040.5. Structural modifications in that class of compounds have revealed a considerable potential for optimisation in respect of the action on microtubules. Outstanding results have been obtained, inter alia, by formal esterification of those broneols with an acid of the Sk-H type. By synthesising the taxol derivatives described herein, in which the isoserine chain of the taxol has been replaced by Sk, the intention was to study whether it is also possible in that class of substances to achieve an improved stabilisation of microtubules, compared with taxol.

The invention relates to pharmacologically active compounds that havethe ability to influence the polymerisation and depolymerisation oftubulin.

A range of natural mitosis toxins are used as anti-tumour agents or arein the process of being clinically tested. There are various classes ofsuch mitosis toxins that either demonstrate their cytotoxic action byinhibiting the polymerisation of microtubules in the spindle system (forexample, Vinca alkaloids, colchicine) or achieve their cytotoxic actionby a GTP-independent increase in the polymerisation of the tubulin andby preventing the depolymerisation of microtubules (for example, taxol,taxoters).

Owing to their physico-chemical properties, hitherto not understood, andas a result of the characterstics of neoplastic cells, mitosis toxinshave a certain selectivity for tumour cells, but there still remains anot inconsiderable cytotoxicity towards non-transformed cells. Thesearch for more selective compounds that are easier to manufactureand--like the taxane class of substances--are able to inhibit thedepolymerisation of microtubules, had, surprisingly, led to thediscovery of borneol esters, as described in P 4416374.6 and 19513040.5.Structural modifications in that class of compounds have revealed aconsiderable potential for optimisation in respect of the action onmicrotubules. Outstanding results have been obtained, inter alia, byformal esterification of those borneols with an acid of the Sk-H type.By synthesising the taxol derivatives described herein, in which theisoserine chain of the taxol has been replaced by Sk, the intention wasto study whether it is also possible in that class of substances toachieve an improved stabilisation of microtubules, compared with taxol.

Further, the following documents disclose compounds showingpharmacological activity:

WO-A-94 21 252

Bioorg. Med. Chem. Lett. (1994) 4(11), 1381-1384

EP-A-0 534 708

Angew. Chem. Int. Ed. Engl. (1994) 33(1), 15-44

WO-A 95 13 053

WO-A-95 19 994

J. Pharm. Sci. (1995) 84(10), 1223-1230

WO-A-96 00 724

However, compounds disclosed in WO-A-94 21 252 form the closest state ofthe art.

Surprisingly, it has now been found that the compounds of formula Iaccording to the invention, compared with taxol and state of the artcompounds, have an advantageously altered activity profile. In additionto a clearly improved stabilisation of microtubules, the compounds offormula I demonstrate an additional influence on the polymerisation oftubulin.

The taxanes according to the invention are characterised by the generalformula I ##STR1## wherein Sk may be ##STR2## R¹ may be hydrogen or C₁-C₁₀ acyl, R² may be α-OH or β-OH,

R³ may be C₁ -C₁₀ alkyl, X-substituted phenyl, C₁ -C₁₀ alkoxy,

X may be hydrogen, halogen, --N₃ or --CN, and

free hydroxy groups in I may be functionally modified further byetherification or esterification,

and also the α-, β- and γ-cyclodextrin clathrates thereof, and alsocompounds of the general formula I encapsulated with liposomes.

There come into consideration as alkyl group R³ straight-chained orbranched alkyl groups having from 1 to 10 carbon atoms, such as, forexample, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl,pentyl, isopentyl, neopentyl, heptyl, hexyl and decyl. Alkyl groupshaving from 1 to 4 carbon atoms are preferred. The acyl and alkoxygroups contained in R¹ and R³, respectively, of general formula Icontain from 1 to 10 carbon atoms, with formyl, acetyl, propionyl andisopropionyl groups, and methoxy, ethoxy, propoxy, isopropoxy andt-butoxy groups, respectively, being preferred.

Halogen in the definition of X is fluorine, chlorine, bromine or iodine.

Preferred compounds of the general formula I are:

3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-taxol,

3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-epi-taxol

3'-desphenyl-3'-(4-pyridyl)-2,3'-N-bisdebenzoyl-3'-N-methoxycarbonyl-taxol,and

3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-10-desacetyl-taxol.

The invention relates also to a process for the preparation of borneolderivatives of formula I, which process is characterised in that analcohol of the general formula II ##STR3## wherein R¹ and R² are asdefined hereinabove and hydroxy groups contained in II are optionallyprotected, is reacted with a compound of the general formula IIIa, IIIbor IIIc, ##STR4## wherein R³ is as defined hereinabove and X' may behydroxy, O-alkyl or halogen, and wherein free hydroxy groups areprotected by etherification or esterification, to form compounds of thegeneral formula I in which free hydroxy groups may be functionallymodified further by etherification or esterification.

For the esterification of the alcohol function in II, deprotonation iscarried out with a base, such as, for example, a metal hydride (forexample sodium hydride), an alkali metal alcoholate (for example sodiummethanolate, potassium tert-butanolate), an alkali metalhexamethyldisilazane (for example sodium hexamethyldisilazane),1,5-diazabicyclo[4.3.0]non-5-ene (DBN),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), triethylamine,4-(dimethylamino)pyridine (DMAP) or 1,4-diazabicyclo[2.2.2]octane(DABCO) and reaction is carried out with a carboxylic acid derivative ofthe general formula III in an inert solvent, such as, for example,dichloromethane, diethyl ether, or tetrahydrofuran, at from -70° C. to+50° C. The preferred reaction is with sodium hydride as base, a cyclicacid amide as carboxylic acid derivative and tetrahydrofuran as solventat temperatures of from -10° C. to +25° C.

Free hydroxy groups in I may be functionally modified further inaccordance with methods known to a person skilled in the art, forexample by etherification or esterification. For example, free hydroxygroups may be converted into pyridinium salts with physiologicallytolerable acids, into phosphates or salts thereof with physiologicallytolerable bases or into esters thereof, into sulphates or salts thereofwith physiologically tolerable bases or into esters thereof, or intoesters and ethers with water-soluble polymers. Ethers and esters ofcompounds that are themselves able to demonstrate tumour-inhibitingaction can also be prepared.

Biological effects and fields of use of the novel taxol derivatives:

The novel compounds of formula I are valuable drugs. They interact withtubulin by influencing the polymerisation of tubulin and stabilisingmicrotubules that have formed and are thus able to influence celldivision in a phase-specific manner. That affects, especially, rapidlygrowing neoplastic cells, the growth of which is largely uninfluenced byintercellular regulating mechanisms. Active ingredients of that typeare, in principle, suitable for the treatment of disorders in whichinfluence on cell division may be indicated therapeutically.

There may be mentioned by way of example the treatment of malignanttumours, malaria, the treatment of disorders caused by gram-negativebacteria, and also the treatment of disorders of the central andperipheral nervous system that are based on excitotoxic mechanisms, suchas, for example, the treatment of acute neurodegenerative symptoms, suchas arise, for example, as a result of stroke or traumatic braininjuries, the treatment of chronic neurodegenerative symptoms includingAlzheimer's disease, and also the treatment of amyotrophic lateralsclerosis.

There may be mentioned as a field of use for malignant tumours, forexample, the treatment of ovarian, stomach, colonic, adeno, breast,lung, head and neck carcinomas, malignant melanoma and acute lymphocyticand myelocytic leukaemia.

The compounds according to the invention may be used generally on theirown or, to obtain additive or synergistic effects, in combination withother active principles and classes of substances that may be used inthe fields of therapy in question.

Taking the example of tumour therapy, there may be mentioned thecombination with

platinum complexes, such as, for example, cisplatin and carboplatin,

intercalating substances, for example, from the class of theanthracyclines, such as, for example, doxorubicin, or from the class ofthe anthrapyrazoles, such as, for example, Cl -941,

substances that interact with tubulin, for example from the class of theVinca alkaloids, such as, for example, vincristine and vinblastine, orfrom the novel class of the borneol esters described in P 4416374.6 and19513040.5, or from the class of the macrolides, such as, for example,rhizoxin, or other compounds, such as, for example, colchicine,combretastatin A-4 and epothilon A and B,

DNA topoisomerase inhibitors, such as, for example, camptothecin,etoposide, topotecan and teniposide,

folate or pyrimidine antimetabolites, such as, for example, lometrexoland gemcitubin,

DNA alkylating compounds, such as, for example, adozelesin anddystamycin A,

growth factor inhibitors (for example, inhibitors of PDGF, EGF, TGFb,EGF), such as, for example, somatostatin, suramin, bombesin antagonists,

inhibitors of tyrosine protein kinase or of the protein kinases A and C,such as, for example, erbstatin, genistein, staurosporine, ilmofosin and8-Cl-cAMP,

anti-hormones from the class of the antigestagens, such as, for example,mifepristone, onapristone or from the class of the anti-oestrogens, suchas, for example tamoxifen, or from the class of the anti-androgens, suchas, for example, cyproterone acetate,

metastasis-inhibiting compounds, for example, from the class of theeicosanoids, such as, for example, PGI₂, PGE₁, 6-oxo-PGE₁ and stablederivatives thereof (for example iloprost, cicaprost, beraprost),

inhibitors of trans-membrane Ca²⁺ influx, such as, for example,verapamil, galopamil, flunarizine, diltiazem, nifedipine and nimodipine,

neuroleptics, such as, for example, chlorpromazine, trifluoperazine,thioridazine and perphenazine,

local anaesthetics, such as, for example, carbanilate-Ca7, cinchocaine,carbanilate-Ca₃, articaine, carbanilate, lidocaine,

angiogenesis-inhibiting substances, such as, for example, anti-VEGFantibodies, endostatin B, interferon a, AGM 1470, and

inhibitors of cell proliferation in psoriasis, Kaposi's sarcoma andneuroblastoma.

The invention relates also to medicaments based on compounds of thegeneral formula I that are pharmaceutically acceptable, that is to saycompounds that are not toxic in the doses used, optionally together withcustomary excipients, carriers and additives.

The compounds according to the invention may be processed in accordancewith methods of galenic pharmacy known per se to pharmaceuticalpreparations for enteral, percutaneous, parenteral or localadministration. They may be administered in the form of tablets,dragees, gelatin capsules, granules, suppositories, implants,injectable, sterile aqueous or oily solutions, suspensions or emulsions,ointments, creams and gels. The invention therefore relates also to theuse of the compounds according to the invention in the preparation ofmedicaments.

The active ingredient(s) may be mixed with excipients that are customaryin galenic pharmacy, such as, for example, gum arabic, talc, starch,mannitol, methylcellulose, lactose, surfactants such as Tweens or Myrj,magnesium stearate, aqueous or non-aqueous carriers, paraffinderivatives, wetting, dispersing, emulsifying and preserving agents, andflavourings to adjust the taste (for example ethereal oils).

The invention therefore relates also to pharmaceutical compositions andmedicaments that contain at least one compound according to theinvention as active ingredient. A unit dose contains approximately from0.1 to 100 mg of active ingredient(s). The dosage of the compoundsaccording to the invention in humans is approximately from 0.1 to 1000mg per day.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the course of the change in absorption as a function oftime and temperature(3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-taxol,Application Example 2).

FIG. 2 shows the course of the change in absorption as a function oftime and temperature(3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-epi-taxol,Application Example 3).

FIG. 3 shows the course of the change in absorption as a function oftime and temperature(3'-desphenyl-3'-(4-pyridyl)-2,3'-N-bisdebenzoyl-3'-N-methoxycarbonyl-taxol,Application Example 4).

FIG. 4 shows the course of the change in absorption as a function oftime and temperature(3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-10-esacetyl-taxol,Application Example 5).

The following Examples serve to explain further the preparation of thecompounds according to the invention.

EXAMPLE 13'-Desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-taxol

8.6 μl of a 1M solution of tetrabutylammonium fluoride intetrahydrofuran are added at 3° C., under an atmosphere of dry argon, toa solution of 3.1 mg (2.9 μmol) of the compound prepared in accordancewith Example 1a in 0.5 ml of anhydrous tetrahydrofuran, and the reactionmixture is stirred for 30 minutes. The mixture is poured into asaturated sodium hydrogen carbonate solution and extraction is carriedout with dichloromethane, the organic extract is concentrated and theresidue is purified by chromatography on one half of an analyticalthin-layer plate. Ethyl acetate is used as mobile phase and a mixture ofdichloromethane and methanol is used as eluting agent. 0.4 mg (0.5 μmol,17%) of the title compound is isolated.

¹ H-NMR (CDCl₃): d=1.16 (3H), 1.25 (3H), 1.70 (3H), 1.75 (1H), 1.84(3H), 1.90 (1H), 2.26 (3H), 2.25-2.38 (2H), 2.38 (3H), 2.48 (1H), 2.56(1H), 3.62 (3H), 3.81 (1H), 4.19 (1H), 4.31 (1H), 4.40 (1H), 4.71 (1H),4.95 (1H), 5.37 (1H), 5.66 (1H, 5.69 (1H), 6.28 (1H), 6.31 (1H), 7.40(2H), 7.51 (2H), 7.61 (1H), 8.11 (2H), 8.66 (2H) ppm.

EXAMPLE 1a2'-Triisopropylsilyl-3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-triethylsilyl-taxol

12 mg of an approximately 60% sodium hydride dispersion are added at 3°C., under an atmosphere of dry argon, to a solution of 4.2 mg (6.0 μmol)and 11.4 mg of the compounds prepared in accordance with Examples 1b and1c in 0.1 ml of anhydrous tetrahydrofuran, and the mixture is heated to30° C. and stirred for 30 minutes. The mixture is cooled again to 3° C.,30% aqueous acetic acid is added and extraction is carried out severaltimes with diethyl ether. The combined organic extracts are washed witha saturated sodium chloride solution and dried over magnesium sulphate.After filtration and removal of solvent, the residue is purified bychromatography on two analytical thin-layer plates. A mixture ofn-hexane and ethyl acetate is used as mobile phase, and a mixture ofdichloromethane and methanol is used as eluting agent. 3.7 mg (3.4 μmol,57%) of the title compound are isolated.

¹ H-NMR (CDCl₃): d=0.60 (6H), 0.80-1.02 (30H), 1.25 (6H), 1.70 (3H),1.91 (1H), 2.03 (3H), 2.14 (1H), 2.20 (3H), 2.36 (1H), 2.49 (3H), 2.53(1H), 3.54 (3H), 3.84 (1H), 4.18 (1H), 4.30 (1H), 4.49 (1H), 4.85 (1H),4.93 (1H), 5.30 (1H), 5.60 (1H), 5.70 (1H), 6.32 (1H), 6.47 (1H), 7.28(2H), 7.49 (2H), 7.59 (1H), 8.13 (2H), 8.64 (2H) ppm.

EXAMPLE 1b 7-Triethylsilyl-baccatin III

21 μl of triethylchlorosilane and 10.3 mg of imidazole are added at 3°C., under an atmosphere of dry argon, to a solution of 3.7 mg (6.3 μmol)of chromatographically purified baccatin III (Calbiochem Corp.) in 0.3ml of anhydrous dimethylformamide, and the reaction mixture is stirredfor one hour. The mixture is poured into a saturated sodium hydrogencarbonate solution, extraction is carried out several times with diethylether followed by washing with a saturated sodium chloride solution, andthe combined organic extracts are concentrated. The residue obtainedafter filtration and removal of solvent is purified by chromatography onone half of an analytical thin-layer plate. A mixture of n-hexane andethyl acetate is used as mobile phase, and a mixture of dichloromethaneand methanol is used as eluting agent 3.0 mg (5.6 μmol, 88%) of thetitle compound are isolated.

¹ H-NMR (CDCl₃): d=0.59 (6H), 0.92 (9H), 1.06 (3H), 1.20 (3H), 1.62(1H), 1.69 (3H), 1.88 (1H), 2.04 (1H), 2.19 (6H), 2.28 (2H), 2.29 (3H),2.53 (1H), 3.88 (1H), 4.14 (1H), 4.31 (1H), 4.50 (1H), 4.83 (1H), 4.98(1H), 5.63 (1H), 6.47 (1H), 7.49 (2H), 7.61 (1H), 8.11 (2H) ppm.

EXAMPLE 1c(3R,4S)-1-(Methoxycarbonyl)-3-triisopropylsilyloxy-4-(4-pyridyl)-2-azetidinone

573 mg of 4-dimethylaminopyridine and 193 μl of chloroformic acid methylester are added at 3° C., under an atmosphere of dry argon, to asolution of 250 mg (0.78 mmol) of the compound prepared in accordancewith Example 1d in 10 ml of anhydrous dichloromethane and the reactionmixture is heated to 23° C. and stirred for a further 16 hours. Themixture is poured into a saturated ammonium chloride solution,extraction is carried out several times with diethyl ether followed bywashing with a saturated sodium chloride solution, and the combinedorganic extracts are concentrated. The residue obtained after filtrationand removal of solvent is purified by chromatography on approximately150 ml of fine silica gel using a mobile phase mixture of n-hexane andethyl acetate. 251 mg (0.66 mmol, 85%) of the title compound areisolated.

¹ H-NMR (CDCl₃): d=0.82-1.07 (21H), 3.82 (3H), 5.11 (1H), 5.26 (1H),7.23 (2H), 8.61 (2H) ppm.

EXAMPLE 1d (3R,4S)-3Triisopropylsilyloxy-4-(4-pyridyl)-2-azetidinone

A solution of 67.3 g of ceric ammonium nitrate in 700 ml of water isadded at 3° C., under an argon atmosphere, to a solution of 17.2 g (40.3mmol) of the compound prepared in accordance with Example 1e in 384 mlof acetonitrile, and the reaction mixture is stirred for 30 minutes. Themixture is poured into a saturated sodium hydrogen carbonate solution,extraction is carried out several times with ethyl acetate, and thecombined organic extracts are washed with a 1% sodium hydroxide solutionand dried over magnesium sulphate. The residue obtained after filtrationand removal of solvent is purified by chromatography on approximately800 ml of fine silica gel using a mobile phase mixture of n-hexane andethyl acetate. 7.89 g (24.6 mmol, 61%) of the title compound areisolated.

¹ H-NMR (CDCl₃): d=0.78-1.07 (21H), 4.81 (1H), 5.23 (1H), 6.39 (1H),7.28 (2H 8.59 (2H) ppm.

EXAMPLE 1e(3R,4S)-1-(4-Methoxyphenyl)-3-triisopropylsilyloxy-4-(4-pyridyl)-2-azetidinone

A solution of 12.6 ml of freshly distilled diisopropylamine in 70 ml ofanhydrous tetrahydrofuran is cooled to -30° C. under an atmosphere ofdry argon, 37.6 ml of a 2.4M solution of n-butyllithium in n-hexane areadded and the mixture is heated to 0° C. After 30 minutes, the mixtureis cooled to -78° C., a solution of 22.1 g (56.6 mmol) of(1R,2S)-2-phenyl-1-cyclohexyl-triisopropylsilyloxyacetate, preparedanalogously to the process described in Tetrahedron Vol. 48, No. 34, pp.6985-7012, 1992, in 70 ml of anhydrous tetrahydrofuran is added dropwisethereto and the mixture is stirred for 3 hours. A solution of 15.6 g(73.5 mmol) of the aldimine prepared in accordance with Example 1f in150 ml of anhydrous tetrahydrofuran is then added and the mixture isheated to 23° C. in the course of 16 hours. The mixture is poured into asaturated ammonium chloride solution, extraction is carried out severaltimes with ethyl acetate followed by washing with a saturated sodiumchloride solution, and the combined organic extracts are concentrated.The residue obtained after filtration and removal of solvent is purifiedby chromatography on approximately 1.8 liters of fine silica gel using amobile phase mixture of n-hexane and ethyl acetate. 17.2 g (40.3 mmol,71%) of the title compound are isolated.

¹ H-NMR(CDCl₃): d=0.82-1.12 (21H), 3.76 (3H), 5.12 (1H), 5.29 (1H), 6.80(2H), 7.19-7.30 (4H), 8.60 (2H) ppm.

EXAMPLE 1f N-(4-Methoxyphenyl)-(4-pyridyl)aldimine

7.8 ml of pyridine-4-aldehyde and 8.4 g of magnesium sulphate are added,under an atmosphere of dry argon, to a solution of 10 g (81.1 mmol) of4-anisidine in 120 ml of anhydrous dichloromethane, and the mixture isstirred at 23° C. for 4 hours. The residue obtained after filtration andremoval of solvent is recrystallised from n-hexane. 15.9 g (74.9 mmol,92%) of the title compound are isolated.

¹ H-NMR (CDCl3₃): d=3.83 (3H), 6.95 (2H), 7.29 (2H), 7.73 (2H), 8.47(1H), 8.73 (2H) ppm.

EXAMPLE 23'-Desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-epi-taxol(A) and3'-desphenyl-3'-(4-pyridyl)-2,3'-N-bisdebenzoyl-3'-N-methoxycarbonyl-taxol(B)

42 μl of a 1M solution of tetrabutylammonium fluoride in tetrahydrofuranare added at 3° C., under an atmosphere of dry argon, to a solution of15 mg (13.9 μmol) of the compound prepared in accordance with Example 1ain 0.5 ml of anhydrous tetrahydrofuran, and the mixture is stirred for30 minutes at 3° C., heated to 23° C. and stirred for a further 30minutes. The mixture is poured into a saturated sodium hydrogencarbonate solution, extraction is carried out with dichloromethane, theorganic extract is concentrated and the residue is purified bychromatography on two analytical thin-layer plates. A mixture of ethylacetate and methanol is used as mobile phase, and a mixture ofdichloromethane and methanol is used as eluting agent.

3.8 mg (4.7 μmol, 34%) of the title compound A, 2.4 mg (3.4 μmol, 25%)of the title compound B and also 1.2 mg (1.5 μmol, 11%) of the compounddescribed in Example 1 are isolated.

¹ H-NMR (CDCl₃) of A: d=1.18 (3H), 1.23 (3H), 1.68 (3H), 1.71 (1H), 1.80(1H), 1.83 (3H), 2.15-2.48 (4H), 2.21 (3H), 2.49 (3H), 3.56 (3H), 3.71(1H), 3.92 (1H), 4.37 (2H), 4.63 (1H), 4.71 (1H), 4.91 (1H), 5.37 (1H),5.67 (1H), 5.76 (1H), 6.34 (1H), 6.81 (1H), 7.33 (2H), 7.51 (2H), 7.61(1H), 8.16 (2H), 8.63 (2H) ppm.

EXAMPLE 33'-Desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-10-desacetyl-taxol

20.4 μl of 4N hydrochloric acid are added, under an argon atmosphere, toa solution of 9.0 mg (10.2 μmol) of compound A prepared in accordancewith Example 3a in 0.8 ml of ethanol and 0.2 ml of tetrahydrofuran, andthe reaction mixture is stirred at 23° C. for one hour. The addition ofhydrochloric acid is repeated a further twice, each time after onehour's stirring, a saturated sodium hydrogen carbonate solution isadded, extraction is carried out with dichloromethane, the organicextract is concentrated and the residue is purified by chromatography ontwo analytical thin-layer plates. A mixture of ethyl acetate and ethanolis used as mobile phase, and a mixture of dichloromethane and methanolis used as eluting agent 6.5 mg (8.5 μmol, 83%) of the title compoundare isolated.

¹ H-NMR (CDCl₃): d=1.13 (3H), 1.24 (3H), 1.78 (3H), 1.83 (3H), 1.73-1.96(3H), 2.25 (2H), 2.37 (3H), 2.60 (1H), 3.62 (3H), 3.92 (1H), 4.14-4.28(2H), 4.20 (1H), 4.32 (1H), 4.69 (1H), 4.94 (1H), 5.21 (1H), 5.36 (1H),5.68 (1H), 5.83 (1H), 6.30 (1H), 7.34 (2H), 7.50 (2H), 7.62 (1H), 8.10(2H), 8.61 (2H) ppm.

EXAMPLE 3a3'-Desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-triethylsilyl-10-desacetyl-taxol(A) and3'-Desphenyl-3'-(4-pyridyl)-2,3'-N-bisdebenzoyl-3'-N-methoxycarbonyl-7-triethylsilyl-10-desacetyl-taxol(B)

25 mg (23 μmol) of the crude product prepared in accordance with Example3b are reacted at -10° C. analogously to Example 1 and, after working upand purification, 9.0 mg (10.2 μmol, 44%) of the title compound A, 2.5mg (3.2 μmol, 14%) of the title compound B and also 2.2 mg (2.9 μmol,12%) of the title compound of Example 3 are isolated.

¹ H-NMR (CDCl₃) of A: d=0.43-0.67 (6H), 0.94 (9H), 1.13 (3H), 1.24 (3H),1.70 (1H), 1.76 (3H), 1.87 (3H), 1.93 (1H), 2.24 (2H), 2.36 (3H), 2.48(1H), 3.62 (3H), 3.87 (1H), 4.18 (1H), 4.29 (1H), 4.34 (1H), 4.68 (1H),4.91 (1H), 5.12 (1H), 5.36 (1H), 5.63 (1H), 5.77 (1H), 6.29 (1H), 7.33(2H), 7.49 (2H), 7.60 (1H), 8.10 (2H), 8.60 (2H) ppm.

¹ H-NMR (CDCl₃) of B: d=0.45-0.64 (6H), 0.94 (9H), 1.02 (3H), 1.25 (4H),1.67 (1H), 1.72 (3H), 1.82 (3H), 1.88-2.12 (2H), 2.18 (4H), 2.47 (1H),3.50 (1H), 3.67 (1H), 3.70 (3H), 3.90 (1H), 4.26 (1H), 4.30 (1H), 4.59(1H), 4.61 (1H), 4.66 (1H), 4.91 (1H), 5.03 (1H), 5.27 (1H), 5.65 (1H),6.25 (1H), 7.32 (2H), 8.62 (2H), ppm.

EXAMPLE 3b2'-Triisopropylsilyl-3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-triethylsilyl-10-desacetyl-taxol

0.23 ml of hydrazinium hydroxide is added, under an argon atmosphere, toa solution of 25 mg (23.2 μmol) of the compound prepared in accordancewith Example 1a in 1.2 ml of ethanol, and the mixture is stirred at 23°C. for 24 hours. The mixture is poured into a saturated sodium chloridesolution, extraction is carried out several times with ethyl acetate anddrying is carried out over magnesium sulphate. The residue obtainedafter filtration and removal of solvent is further reacted without beingpurified. 22 mg (max. 21 μmol, max. 91%) of the title compound, whichstill contains small amounts of starting material, are isolated.

The following Application Examples substantiate the biological activityof the compounds according to the invention without limiting theirapplication to those Examples.

Application Example 1 Isolation and Purification of Tubulin

Bovine brains (each 330 g) are removed from freshly opened cows' headsand are transported in ice-cooled PM4-M buffer. Each brain is freed ofthe meninges and also of any thrombi and is homogenised with sufficientPM4M buffer in a cooling chamber. The homogenised material from 2 bovinebrains is made up to a total volume of 1.0 liter with a total of 500 mlof buffer and is subjected to a first centrifugation (GSA rotor, 15minutes, 4° C., 6500 g). The supernatant is freed of the sebaceous skinon the surface, filtered over 4 layers of thin muslin, transferred intocounter-balanced centrifuge tubes (420 ml) and centrifuged again (Ti 45rotor, 96000 g, 75 minutes, 4° C.). The supernatant is removed bypipette through the pellet and filtered over 6 layers of thin muslin,and a 50 mM GTP solution in 0.001M bicarbonate/PBS is added to obtain afinal concentration of 1 mM. A first polymerisation is carried out incounter-balanced centrifuge tubes for 45 minutes in a water bath warmedto 37° C. The microtubules that have formed are removed bycentrifugation (Ti 45 rotor, 27° C., 96000 g, 60 minutes), thesupernatant is carefully removed by pipette and the very soft,opalescent pellet is carefully separated from the wall with a spatula.40 ml of cold PM buffer are then added to the pellet, followed byhomogenisation using a small glass mortar and incubation overnight (from12 to 16 hours) on ice in counter-balanced centrifuge tubes in a coolingchamber. The depolymerisation product is removed by centrifugation inthe Ti 60 rotor (4° C.; 96000 g, 60 minutes) and the supernatant isdiluted with PM8-M buffer to 1:1, incubated in counter-balancedcentrifuge tubes at 37° C. for 45 minutes and centrifuged again (Ti 45rotor, 27° C., 96000 g, 60 minutes). The supernatant is carefullyremoved by pipette and the very soft opalescent pellet is taken up in 20ml of cold PM buffer, homogenised carefully using a small glass mortarand incubated on ice for 30 minutes. The renewed centrifugation (Ti 60rotor, 4° C., 96000 g, 60 minutes) gives tubulin, the protein content ofwhich is determined according to Pearce or according to a photometricmeasurement at 280 nm. In the protein determination, dilutions ofisolated material to PM buffer of 1:10, 1:20 and 1:40 are used. The PMbuffer has own extinction and is deducted as a zero value from thedetermined protein content. The isolated material is diluted with PMbuffer to the intended protein concentration (2 mg/ml).

Application Example 2 Biological effect on tubulin of3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-taxol

The measurement of the polymerisation of tubulin and thedepolymerisation of microtubules is carried out by photometry. Beforethe measurement, the tubulin prepared in accordance with ApplicationExample 1 is thawed and degassed for 15 minutes. The photometer is setto a wavelength of 350 nm. 3 μl of solvent/sample, 6 μl of GTP (0-25μmol/l final.) and 291 μl of tubulin (2 mg protein/ml) are pipetted intothe dry and cleaned dishes (10 mm). The sample is carefully stirred(without causing air bubbles), immediately placed in dish carriages andthe measuring operation is started at 37° C. Once the polymerisationmaximum has been reached (solvent control and taxol 1E-5 mol/l after 20minutes), the depolymerisation is initiated by reducing the temperatureto 15° C. The measuring operation is stopped at the end of thedepolymerisation and the course of the change in absorption isrepresented in graph form as a function of time and temperature (seeFIG. 1).

The Figure clearly shows that taxol speeds up the polymerisation oftubulin in comparison with the control and inhibits depolymerisation,whilst the compound according to the invention3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-taxolinhibits the polymerisation and stabilises the microtubules that haveformed distinctly better than taxol.

Application Example 3 Biological effect on tubulin of3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-epi-taxol

3'-Desphenyl-3'-(4pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-epi-taxolcauses a much more pronounced acceleration of the polymerisation oftubulin and stabilises the microtubules that have formed very muchbetter than taxol. The results are shown in FIG. 2.

Application Example 4 Biological effect on tubulin of3'-desphenyl-3'-(4-pyridyl)-2,3'-N-bisdebenzoyl-3'-N-methoxycarbonyl-taxol

3'-Desphenyl-3'-(4-pyridyl)-2,3'-N-bisdebenzoyl-3'-N-methoxycarbonyl-taxolcauses a much more pronounced acceleration of the polymerisation oftubulin and stabilises the microtubules that have formed better thantaxol. The results are shown in FIG. 3.

Application Example 5 Biological effect on tubulin of3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-10-desacetyl-taxol

3'-Desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-10-desacetyl-taxoldoes not differ from taxol in respect of the polymerisation behaviour oftubulin, but it does stabilise the microtubules that have formedsubstantially better than taxol. The results are shown in FIG. 4.

Application Example 6 Anti-proliferative effect on tumour cell lines of:3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-taxol(1),3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-epi-taxol(2),3'-desphenyl-3'-(4-pyridyl)-2,3'-N-bisdebenzoyl-3'-N-methoxycarbonyl-taxol(3) and3'-desphenyl-3'(pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-10-desacetyl-taxol(4)

MDA MB 435 mammary carcinoma cells (5000 cells/well) are placed inmicrotitre plates (day 0, RPMI medium, 1% non-essential amino acids, 1%pyruvate, 10% foetal calf serum). The addition of the substance inseveral concentrations is carried out on day 1. The anti-proliferativeeffect is determined on day 3 using the MTT method. The IC₅₀ values aredetermined therefrom. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                      MDA-MB 435 cells                                                Compound      IC.sub.50 [nM]                                                  ______________________________________                                        1             2.0                                                             2             0.8                                                             3             8000                                                            4             60                                                              Taxol         3                                                               (reference)                                                                   ______________________________________                                    

3'-Desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-taxoldemonstrates an activity similar to that of taxol, whilst3'-desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-epi-taxoldemonstrates a significantly improved inhibiting activity compared withtaxol.

What is claimed is:
 1. A taxane of the formula I: ##STR5## wherein Sk is##STR6## R¹ is hydrogen or C₁ -C₁₀ acyl, R² is α-OH,R³ is C₁ -C₁₀ alkyl,X-substituted phenyl, or C₁ -C₁₀ alkoxy, X is hydrogen, halogen, --N₃ or--CN, or a physiologically acceptable salt thereof, or phosphate orsulfate ester thereof, or ester or ether thereof with a physiologicallyacceptable water-soluble polymer, or a α-, β- or γ-cyclodextrinclathrate thereof. 2.3'-Desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-7-epi-taxolaccording to claim
 1. 3.3'-Desphenyl-3'-(4-pyridyl)-3'-N-debenzoyl-3'-N-methoxycarbonyl-10-desacetyl-taxolaccording to claim
 1. 4. Process for the preparation of taxanederivatives of the formula I according to claim 1, wherein an alcohol ofthe formula II ##STR7## wherein R¹ and R² are as defined hereinabove andhydroxy groups contained in II are optionally protected, is reacted witha compound of the general formula IIa, IIIb or IIIc, ##STR8## wherein R³is as defined hereinabove and X' may be hydroxy, O-alkyl or halogen, andwherein free hydroxy groups are protected by etherification oresterification, to form compounds of the general formula I in which freehydroxy groups may be functionally modified further by etherification oresterification.
 5. A taxane of the formula I: ##STR9## wherein Sk is##STR10## R¹ is hydrogen or C₁ -C₁₀ acyl, R² is α-OH,R³ is C₁ -C₁₀alkyl, X-substituted phenyl, or C₁ -C₁₀ alkoxy, X is halogen, --N₃ or--CN, or a physiologically acceptable salt thereof, or phosphate orsulfate ester thereof, or a α-, β- or γ-cyclodextrin clathrate thereof.6. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 7. A method of inhibiting thegrowth of neoplastic cells comprising administering to a patient in needthereof, an effective amount of a compound of claim
 1. 8. A method ofclaim 7 comprising treating a patient in need thereof who is sufferingfrom an ovarian, stomach, colonic, adeno, breast, lung, head or neckcarcinoma, a malignant melanoma or acute lymphocytic or myelocyticleukemia.
 9. A pharmaceutical composition of claim 6 wherein saidcompound is encapsulated within a liposome.